1521-0111/84/3/384–392$25.00
MOLECULAR PHARMACOLOGY
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/mol.113.086348
Mol Pharmacol 84:384–392, September 2013
Novel Amino-Carbonitrile-Pyrazole Identified in a Small Molecule
Screen Activates Wild-Type and ΔF508 Cystic Fibrosis
Transmembrane Conductance Regulator in the Absence of
a cAMP Agonist
Wan Namkung, Jinhong Park, Yohan Seo, and A. S. Verkman
Departments of Medicine and Physiology, University of California, San Francisco, California (W.N., A.S.V.); and College of
Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, South Korea (W.N., J.P., Y.S.)
ABSTRACT
Cystic fibrosis (CF) is caused by loss-of-function mutations in
the CF transmembrane conductance regulator (CFTR) Cl2
channel. We developed a phenotype-based high-throughput
screen to identify small-molecule activators of human airway
epithelial Ca21-activated Cl2 channels (CaCCs) for CF therapy.
Unexpectedly, screening of ∼110,000 synthetic small molecules
revealed an amino-carbonitrile-pyrazole, Cact-A1, that activated
CFTR but not CaCC Cl2 conductance. Cact-A1 produced large
and sustained CFTR Cl2 currents in CFTR-expressing Fisher rat
thyroid (FRT) cells and in primary cultures of human bronchial
epithelial (HBE) cells, without increasing intracellular cAMP and
in the absence of a cAMP agonist. Cact-A1 produced linear
whole-cell currents. Cact-A1 also activated DF508-CFTR Cl2
currents in low temperature-rescued ΔF508-CFTR-expressing
FRT cells and CF-HBE cells (from homozygous ΔF508 patients)
Introduction
Cystic fibrosis (CF), the most common lethal genetic disease
in the Caucasian population, is caused by mutations in the
cystic fibrosis transmembrane conductance regulator (CFTR)
that impair its function as an epithelial cell Cl2 channel in
the airways, pancreas, intestine, and other organs (Quinton,
1983; Wine, 1995; Riordan, 2008; Lukacs and Verkman,
2012). Several new approaches for drug therapy of CF have
emerged, including Ivacaftor [VX-770; N-(2,4-di-tert-butyl-5hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide],
a CFTR potentiator approved to treat CF patients with
the G551D-CFTR mutation (Davis, 2011). Clinical trials of
Ivacaftor in CF patients with the G551D mutation on at least
This work was supported by the Yonsei University Research Fund of 2012
and Basic Science Research Program through the National Research Foundation
of Korea (NRF) funded by the Ministry of Education, Science, and Technology
[NRF-2012R1A1A1040142]; and by the National Institutes of Health National
Institute of Diabetes and Digestive and Kidney Diseases [Grant DK72517].
dx.doi.org/10.1124/mol.113.086348.
in the absence of a cAMP agonist, and showed additive effects
with forskolin. In contrast, N-(2,4-di-tert-butyl-5-hydroxyphenyl)4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770) and genistein produced little or no ΔF508-CFTR Cl2 current in the absence
of a cAMP agonist. In FRT cells expressing G551D-CFTR and in
CF nasal polyp epithelial cells (from a heterozygous G551D/
Y1092X-CFTR patient), Cact-A1 produced little Cl2 current by
itself but showed synergy with forskolin. The amino-carbonitrilepyrazole Cact-A1 identified here is unique among prior CFTRactivating compounds, as it strongly activated wild-type and
ΔF508-CFTR in the absence of a cAMP agonist. Increasing
ΔF508-CFTR Cl2 conductance by an “activator,” as defined by
activation in the absence of cAMP stimulation, provides a novel
strategy for CF therapy that is different from that of a “potentiator,”
which requires cAMP elevation.
one CFTR allele showed improvements in lung function,
pulmonary exacerbations, patient-reported respiratory symptoms, body weight, and sweat chloride concentration (Ramsey
et al., 2011; Davis et al., 2012).
There is continued interest in the development of activators
of wild-type (WT) CFTR, for potential therapy of constipation
and some chronic pulmonary diseases, and of ΔF508-CFTR,
the most common CF-causing CFTR mutation (Lacy and
Levy, 2007; Verkman and Galietta, 2009). Prior studies have
revealed multiple chemical classes of CFTR activators that
likely function by direct interaction with CFTR, including
flavones/isoflavones, benzo[c]quinoliziniums, xanthines, benzimidazolones, fluorescein derivatives, and benzoflavones
(Illek et al., 2000; Galietta et al., 2001; Ma et al., 2002b; Caci
et al., 2003; Springsteel et al., 2003). Various ΔF508-CFTR
“potentiators” have been identified, including VX-770, phenylglycines, sulfonamides, and tetrahydrobenzothiophenes (Yang
et al., 2003; Pedemonte et al., 2005; Van Goor et al., 2009).
ABBREVIATIONS: BrdU, bromodeoxyuridine; CaCC, Ca21-activated Cl2 channel; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane
conductance regulator; FRT, fisher rat thyroid; HBE, human bronchial epithelial; IBMX, 3-isobutyl-1-methylxanthine; PBS, phosphate-buffered
saline; TMEM16A, transmembrane protein 16A; VX-770, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide; VX-809,
3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid; WT, wild-type; YFP, yellow fluorescent
protein.
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Received March 16, 2013; accepted June 19, 2013
Novel Small-Molecule CFTR Activator
Materials and Methods
Forskolin, genistein, and other chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich (St. Louis, MO). VX-770
was purchased from Selleck Chemicals (Houston, TX). CFTRinh-172
was synthesized as described elsewhere (Ma et al., 2002a). Cact-A1 was
purchased from ChemDiv (San Diego, CA). The compound collections
used for screening included ∼100,000 synthetic small molecules
from ChemDiv and Asinex (San Diego, CA), and ∼7500 purified
natural products from Analyticon (Potsdam, Germany), Timtek (Newark,
NJ), and Biomol (Plymouth Meeting, PA). Compounds were maintained as dimethylsulfoxide stock solutions. The HCO32-buffered
solution contained (in mM): 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10
2
D -glucose, 5 HEPES, and 25 NaHCO 3 (pH 7.4). In the half-Cl
solution 65 mM NaCl in the HCO32-buffered solution was replaced
by Na gluconate.
Cell Culture. Calu-3 cells were maintained in Dulbecco’s modified Eagle’s medium/F-12 (1:1) medium containing 10% fetal bovine
serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Calu-3
cells were stably transfected with the halide sensor yellow fluorescent
protein (YFP)-H148Q/I152L/F46L. Fisher rat thyroid (FRT) cells
expressing human WT-, DF508-, and G551D-CFTR, and TMEM16A
(abc) were grown in F-12 Modified Coon’s medium supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and
100 mg/ml streptomycin. Primary cultures of non-CF and CF human
airway epithelial cells were grown at an air-liquid interface as described (Levin et al., 2006). Cells were plated at a density of 5 105
per cm2 onto 12-mm diameter, 0.4-mm pore polycarbonate cell culture
inserts (Snapwell; Corning, Lowell, MA) precoated with human
placental collagen (15 mg/cm2; Sigma-Aldrich). Cultures were grown
at an air-liquid interface in air-liquid interface (ALI) medium at 37°C
in 5% CO2/95% air (Fulcher et al., 2005). The medium was changed
every 2–3 days. Cultures were used 21–30 days after plating, at which
time transepithelial resistance was 400–1000 Ohm/cm2 and an ASL
film was seen.
Cell-Based High-Throughput Screening. Calu-3-YFP cells
were plated in 96-well black-walled microplates (Corning Inc.,
Corning, NY) at a density of 20,000 cells per well in F-12 medium
supplemented with 10% fetal bovine serum, 100 units/ml penicillin
and 100 mg/ml streptomycin. To increase CaCC current, interleukin-4
(10 ng/ml) was added at 24 hours after plating, and the cells were
incubated for an additional 48 hours. Assays were done using an
automated screening platform equipped with Infinite F500 and
Infinite M1000 PRO fluorescence plate readers (Tecan, Durham,
NC). Each well of a 96-well plate was washed 3 times in phosphatebuffered saline (PBS) (200 ml/wash), leaving 50 ml PBS including
10 mM CFTRinh-172. Test compounds (0.5 ml) were added to each well
Fig. 1. Phenotype screen for identification of smallmolecule Cl2 channel activators in Calu-3 cells. (A)
Fluorescence micrograph image of Calu-3 cells stably
expressing the halide-sensitive cytoplasmic fluorescent
sensor YFP-H148Q/I152L/F46L. (B) Time course of YFP
fluorescence after extracellular I2 addition. As indicated, solutions contained vehicle, CFTRinh-172 (10 mM)
with forskolin (10 mM) or ionomycin (1 mM), and
forskolin (10 mM), (mean 6 S.E., n = 4). (C) Screening
protocol. Calu-3 cells stably expressing the halidesensitive YFP were preincubated with CFTRinh-172
and then incubated for 10 minutes with test compound.
Fluorescence was monitored in response to addition of
iodide. (D) Fluorescence measured in single wells of 96well plates, showing examples of inactive and active
compounds.
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Potentiators require a cAMP agonist to increase ΔF508-CFTR
Cl2 current.
The original goal of this study was to perform a highthroughput screen to identify small-molecule activators of
Ca21-activated Cl2 channels (CaCCs) in human airway epithelium. We previously reported small-molecule activators of
a CaCC, transmembrane protein 16A (TMEM16A) (Namkung
et al., 2011b); however, TMEM16A is expressed at low levels
in unstimulated airway epithelium (Namkung et al., 2011a)
and hence is not a good target for CF therapy. We therefore
developed a phenotype screen to identify activators of nonTMEM16A CaCC(s) in the human epithelial cell line Calu-3.
Calu-3 cells transfected with the yellow fluorescent protein–
based halide indicator were incubated with test compounds
and subjected to an inwardly directly I2 gradient. In an
attempt to select for activators of non-CFTR Cl2 channels, the
screen was done in the presence of the thiazolidinone CFTR
inhibitor CFTRinh-172. Unexpectedly, one of the strongest activators of I2 influx, the amino-carbonitrile-pyrazole Cact-A1,
was found to be a CFTR activator that competed with
CFTRinh-172 and activated CFTR in a cAMP-independent
manner, and, unlike prior CFTR activators, without the
requirement of basal CFTR activation in primary cultures of
human airway epithelial cells. Also, unlike prior potentiators
including VX-770, Cact-A1 also activated DF508-CFTR in
human CF airway epithelial cells without a cAMP agonist.
The unique CFTR activation mechanism of Cact-A1 suggests
a novel therapeutic alternative in CF caused by the ΔF508
mutation—an “activator” rather than “potentiator.”
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1 Tris-ATP, and 10 HEPES (pH 7.2). Pipettes were pulled from
borosilicate glass and had resistances of 3–5 MV after fire polishing.
Seal resistances were between 3 and 10 GV. After establishing the
whole-cell configuration, CFTR was activated by forskolin and/or
Cact-A1. Whole-cell currents were elicited by applying hyperpolarizing
and depolarizing voltage pulses from a holding potential of 0 mV to
potentials between 280 and 180 mV in steps of 20 mV. Recordings
were made at room temperature using an Axopatch-200B (Axon Instruments). Currents were digitized with a Digidata 1440A converter
(Axon Instruments), filtered at 5 kHz, and sampled at 1 kHz.
Intracellular Calcium Measurement. FRT cells expressing
human TMEM16A were cultured in 96-well black-walled microplates
and loaded with Fluo-4 NW per the manufacturer’s protocol (Invitrogen,
Carlsbad, CA). Fluo-4 fluorescence was measured with a FLUOstar
Optima fluorescence plate reader equipped with syringe pumps and
custom Fluo-4 excitation/emission filters (485/538 nm).
cAMP Assay. Human bronchial epithelial (HBE) cells grown on
permeable supports and FRT cells grown on 12-well culture plates
were washed 3 times with PBS at 37°C and then incubated in PBS at
37°C containing 100 mM 3-isobutyl-1-methylxanthine (IBMX) for 5
minutes in the absence or presence of forskolin/Cact-A1. After 10minutes incubation, the cells were washed with cold PBS and cytosolic
cAMP was measured using a cAMP immunoassay kit (Parameter
cAMP Immunoassay Kit; R&D Systems, Minneapolis, MN) according
to the manufacturer’s protocol.
Cell Proliferation Assays. Calu-3 cells (human lung epithelial
cells) were seeded (5000 cells/well) on 96-well black-walled microplates. After 24 hours of incubation, cells were treated with different
concentrations of Cact-A1 (0, 3, 10, 30 mM) and then incubated for 2
days. The culture medium was changed every 12 hours, and the cells
Fig. 2. Chemical structures of CFTR activators.
(A) Structures shown of the most potent CFTR
activator of each of four classes (Cact-X1, where
X = A, B, C, or D). (B) Short-circuit (apical
membrane) current measured in WT-CFTR expressing FRT cells in the presence of a transepithelial chloride gradient and after basolateral
membrane permeabilization. Representative current traces showing forskolin (20 mM), Cact-A1,
Cact-B1, Cact-C1, or Cact-D1 stimulated CFTR Cl2
current. Cl2 current was inhibited by addition of
20 mM CFTRinh-172.
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at a 25 mM final concentration. After 5 minutes, 96-well plates were
transferred to a plate reader preheated to 37°C for fluorescence assay.
Each well was assayed individually for CaCCs-mediated I2 influx by
recording fluorescence continuously (200 millisecond per point) for 2
seconds (baseline), then 50 ml of 140 mM I2 solution was added at 2
seconds and then YFP fluorescence was recorded for 6 seconds. The
initial rate of I2 influx following each of the solution additions was
computed from fluorescence data by nonlinear regression.
Short-Circuit Current. Snapwell inserts containing CFTRexpressing FRT or primary culture of human airway cells were
mounted in Ussing chambers (Physiologic Instruments, San Diego,
CA). Forskolin, genistein, VX-770, CFTRinh-172, and Cact-A1 were
added to the apical and basolateral bath solution. For primary
cultures of human airway cells, symmetrical HCO32-buffered solutions were used, and epithelial sodium channel (ENaC) was inhibited
by pretreatment with amiloride (100 mM). For FRT cells, the apical
bath was filled with a half-Cl2 solution, and the basolateral bath was
filled with HCO32-buffered solution, and the basolateral membrane
was permeabilized with 250 mg/ml amphotericin B. All cells were
bathed for a 10-minute stabilization period and aerated with 95%
O2/5% CO2 at 37°C, except for FRT cells expressing WT-CFTR, which
were bathed at room temperature. Apical membrane current (for
FRT cells) and short-circuit current were measured using an
EVC4000 Multi-Channel V/I Clamp (World Precision Instruments,
Sarasota, FL) and recorded using PowerLab/8sp (AD Instruments,
Castle Hill, NSW, Australia).
Patch-Clamp. Whole-cell patch-clamp recordings were performed
on CFTR-expressing FRT cells. The bath solution contained (in mM):
140 NMDG-Cl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4).
The pipette solution contained (in mM): 130 CsCl, 0.5 EGTA, 1 MgCl2,
Novel Small-Molecule CFTR Activator
were treated with Cact-A1. To assess cell proliferation, methanethiosulfonate and bromodeoxyuridine (BrdU) assays were done using
CellTiter 96 AQueous One Solution Cell Proliferation Assay kit
(Promega, Fitchburg, WI) and Cell Proliferation Enzyme-Linked
Immunosorbent Assay, BrdU (colorimetric) kit (Roche Applied Science,
Indianapolis, IN), respectively.
Results
screening to identify CaCC activators, the Calu-3 cells were
preincubated with CFTRinh-172 and test compounds in PBS
before addition of an I2-containing solution (Fig. 1C). I2
addition produced little YFP fluorescence quenching in the
absence of the activators because of the low basal I2 permeability
of Calu-3 cells. Examples of original screening data are shown in
Fig. 1D.
Screening of ∼110,000 small synthetic molecules and natural products yielded 46 compounds that at 10 mM increased
I2 influx by 70% or more compared with that produced by
ionomycin. Secondary testing of the 46 compounds by shortcircuit current measurement in interleukin-4–treated Calu-3
cells showed that nine of compounds increased the Cl2 current,
of which five weakly activated the native CaCC Cl2 current and,
unexpectedly, four strongly activated CFTR Cl2 current, even
in the absence of a cAMP agonist (Fig. 2). The four CFTR
activators, which were identified in the primary screen done
in the presence of 5 mM CFTRinh-172, probably compete with
CFTRinh-172 for binding to CFTR. Of the four CFTR activators, Cact-A1 was further studied because it most strongly
activated CFTR-dependent Cl2 current in primary cultures of
human bronchial epithelial cells.
Fig. 3. Characterization of Cact-A1, a small-molecule
CFTR activator. (A) Cact-A1 concentration-dependent
activation of CFTR in FRT cells expressing human
WT-CFTR (mean 6 S.E., n = 4–7). (B) Cact-A1
reversibility. Cact-A1 (10 mM) addition followed by
washout and readdition. The dashed line shows
Cact-A1-induced Cl2 current without washing. (C)
CFTRinh-172 dose-response for inhibition of CFTR
chloride current in FRT cells expressing WT-CFTR.
CFTR was stimulated by 20 mM forskolin (closed
circles) or 30 mM Cact-A1 (open circles). (D) Shortcircuit current (left) and intracellular calcium concentration (right) measured in FRT cells expressing
TMEM16A. TMEM16A was activated by 100 mM
ATP. (E) Short-circuit current measured in HBE
cells. (Left) CFTR was activated by 20 mM forskolin
and inhibited by 20 mM CFTRinh-172. (Right) CFTR
was activated by indicated concentrations of Cact-A1.
The epithelial sodium channel (ENaC) was inhibited
by pretreatment with 10 mM amiloride. (F) cAMP
accumulation in FRT and HBE cells in response to
addition of Cact-A1 (10 mM) and forskolin (10 mM)
(mean 6 S.E., n = 3–4). (G) Calu-3 cells were treated
with the indicated concentrations of Cact-A1, and
cell proliferation was measured after 2 days using
methanethiosulfonate (MTS) and BrdU assays
(mean 6 S.E., n = 6).
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Identification of CFTR Activators. A cell-based phenotype
screen was developed to identify activators of Ca21-activated
Cl2 channels in human airway epithelia. Cl2 channel activity
was measured in Calu-3 cells, a human airway epithelial
cell line that natively expresses CFTR and CaCC(s) (Wine et al.,
1994; Namkung et al., 2011a). Calu-3 cells were stably transfected with the genetically encoded I2-sensing fluorescent
protein, YFP-F46L/H148Q/I152L, yielding highly fluorescent
cells (Fig. 1A). Using a fluorescence plate reader assay in which
I2 was added to cells after preincubation with agonists/
inhibitors, the CFTR inhibitor CFTRinh-172 blocked forskolinstimulated, CFTR-dependent I2 influx, but not ionomycininduced I2 influx, which involves CaCC activation (Fig. 1B). For
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epithelial cells without elevation of intracellular cAMP and
without the need for a cAMP agonist.
To further characterize CFTR activation by Cact-A1, the
effect of low concentrations of forskolin on Cact-A1-induced
CFTR activation was studied, recognizing that most CFTR
activators, including flavones and benzimidazolones, require
a basal level of CFTR phosphorylation for activation (Galietta
et al., 2001). Figure 4, A–C, shows additive effects of submaximal Cact-A1 and forskolin. In FRT cells expressing
WT-CFTR, a high concentration of VX-770 (30 mM) produced
little CFTR Cl2 current, whereas Cact-A1 strongly activated
CFTR Cl2 current (Fig. 4D). Whole-cell patch-clamp measurements showed that CFTR activation by maximal Cact-A1
produced a linear current/voltage relationship, similar in
magnitude to that produced by maximal forskolin activation
(Fig. 4E).
Cact-A1 Activates and Potentiates DF508-CFTR. A
unique property of Cact-A1 was its ability to activate DF508CFTR Cl2 current in the absence of forskolin, which was
demonstrated in DF508-CFTR expressing FRT and CF-HBE
cells. In these studies, ΔF508-CFTR was rescued by 24-hours’
incubation at low temperature to promote plasma membrane
targeting. In FRT cells, maximal Cact-A1 produced Cl2
current comparable to that of maximal forskolin, which was
∼50% of that produced by maximal Cact-A1 and forskolin
Fig. 4. Additive effects of submaximal Cact-A1 and
forskolin for activation of WT-CFTR. (A and B) Shortcircuit current measured in FRT cells expressing
WT-CFTR. CFTR was activated by indicated concentrations of Cact-A1 and forskolin. CFTR current was
inhibited by 20 mM CFTRinh-172. (C) CFTR activation
with indicated combinations of Cact-A1 and forskolin
(mean 6 S.E., n = 3–5), with 100% inactivation defined
as that produced by 20 mM forskolin alone. (D) Shortcircuit current measured in FRT cells expressing
WT-CFTR. CFTR was stimulated by VX-770, and Cact-A1
and 20 mM forskolin. (E) Whole-cell CFTR Cl2 currents
were recorded at a holding potential at 0 mV, and
pulsing to voltages between 680 mV (in steps of 20 mV)
in FRT cells expressing WT-CFTR. (Right) Summary of
current density data measured at +80 mV (mean 6
S.E., n = 4–6).
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Cact-A1 Reversibly Activates WT-CFTR in Human
Bronchial Epithelial Cells. Short-circuit current measurements in CFTR-expressing FRT cells gave an EC50 of ∼1.6 mM
for Cact-A1 (Fig. 3A). Figure 3B shows that Cact-A1 activation
is reversible, as anticipated. Figure 3C shows a CFTRinh-172
concentration-dependence for inhibition of CFTR Cl2 current
in FRT cells expressing WT-CFTR, comparing the results
for stimulation by forskolin versus Cact-A1. The IC50 for
CFTRinh-172 inhibition was 2.6-fold greater when stimulated
by Cact-A1 (IC50 0.37 mM) than with forskolin (IC50 0.14 mM).
To investigate whether Cact-A1 affects TMEM16A, a CaCC,
or intracellular calcium concentration, short-circuit current,
and intracellular calcium concentration were measured in
FRT cells expressing human TMEM16A. Cact-A1 did not alter
TMEM16A function or intracellular calcium concentration
(Fig. 3D). Short-circuit current measurements in HBE cells
showed strong CFTR activation by Cact-A1, which was inhibited by CFTRinh-172 (Fig. 3E). The activation of CFTR
produced by maximal Cact-A1 in the absence of forskolin was
comparable to that produced by maximal forskolin. Figure 3F
shows that Cact-A1 did not increase intracellular cAMP
concentration in FRT cells or in primary cultures of HBE
cells. Cact-A1 at up to 30 mM showed no cytotoxicity as measured by methanethiosulfonate and BrdU assays (Fig. 3G).
Cact-A1 thus reversibly activates CFTR in human airway
Novel Small-Molecule CFTR Activator
cultures (Fig. 5C). Whole-cell patch-clamp in DF508-CFTR
expressing FRT cells showed that Cact-A1 strongly potentiated forskolin-induced Cl2 current without altering the linear
current/voltage relationship of activated DF508-CFTR (Fig.
5D).
C act -A1 Also Activates and Potentiates G551DCFTR. Cact-A1 activation of the CF-causing gating mutant
G551D-CFTR was investigated in G551D-CFTR expressing
FRT cells. Cact-A1 (30 mM) and forskolin (20 mM), each alone,
produced weak activation of G551D-CFTR (Fig. 6, A and B).
However, combined application of Cact-A1 and forskolin showed
synergistic activation of G551D-CFTR, albeit much smaller
than the potentiation effect of VX-770. VX-770 (10 mM)
strongly increased both forskolin- and Cact-A1-stimulated
G551D-CFTR Cl2 current. Interestingly, Cact-A1 potentiated
the G551D-CFTR Cl2 current activated by maximal forskolin
with VX-770 (Fig. 6A, top left). Figure 6C shows whole-cell
patch-clamp recordings in G551D-CFTR expressing FRT cells.
VX-770 strongly potentiated Cact-A1- or forskolin-induced
Fig. 5. Cact-A1 activates and potentiates lowtemperature rescued DF508-CFTR. (A) Shortcircuit current measured in FRT cells expressing
human DF508-CFTR. DF508-CFTR was rescued by low temperature (27°C) incubation for
24 hours. Where indicated, additions were done
of 30 mM Cact-A1, 20 mM forskolin and 10 mM
VX-770. (B) (Left) Cact-A1 (30 mM), genistein
(50 mM) or VX-770 (10 mM) were added, followed
by forskolin (20 mM). (Right) Summary of shortcircuit current increases (ΔIsc, mean 6 S.E.,
n = 4–6). *P , 0.05; **P , 0.001. (C) (Left)
Short-circuit current in CF-HBE cells from
DF508 homozygous CF patients. Following
low-temperature rescue, DF508-CFTR was stimulated by Cact-A1 and forskolin and then inhibited by 20 mM CFTRinh-172. The bar graph
summarizes short-circuit current data (mean 6
S.E., n = 3–5). (Right) Cact-A1 concentrationdependent activation of DF508-CFTR in CF-HBE
cells (mean 6 S.E., n = 3). (D) Whole-cell DF508CFTR currents were recorded at a holding potential at 0 mV, and pulsing to voltages between
680 mV (in steps of 20 mV) in DF508-CFTR
expressing FRT cells. (Right) Summary of current
density data measured at 180 mV (mean 6 S.E.,
n = 4–6).
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together (Fig. 5A). Interestingly, similar current was seen
with Cact-A1 and VX-770 in the absence of forskolin. Figure 5B
shows measurements done with different combinations of
Cact-A1, forskolin, VX-770, and genistein. Maximal VX-770
(gray line) and genistein (dashed line), each alone, produced
little DF508-CFTR activation in the absence of forskolin
pretreatment, whereas Cact-A1 alone (black line) produced
strong activation. Combined application of Cact-A1, genistein,
or VX-770 with forskolin significantly activated DF508-CFTR
compared with forskolin alone. Therefore, in contrast to the
potentiator VX-770, Cact-A1 functions as both a ΔF508-CFTR
activator (effective without a cAMP agonist) and potentiator
(effective with a cAMP agonist). Similar measurements were
done in HBE cells cultured from airways of DF508 homozygous patients. As found in FRT cells expressing ΔF508-CFTR,
Cact-A1 not only activated DF508-CFTR in the absence of a
cAMP agonist, but also potentiated forskolin-induced Cl2
current by more than 2-fold. The EC50 of Cact-A1 for activation
of DF508-CFTR was 3.5 mM in the primary CF-HBE cell
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Cl2 current. Cact-A1 significantly increased G551D-CFTR
current produced by maximal forskolin and VX-770, in
agreement with the short-circuit current data.
We also measured short-circuit current in primary cultures
of human nasal polyp epithelial cells generated from a compound heterozygous CF patient with G551D/Y1092X-CFTR
mutations. The Y1092X mutation is a nonsense mutation
that does not produce functional protein (Bozon et al., 1994).
Figure 7 shows significant synergy between Cact-A1 and
forskolin, with the maximal current produced by VX-770 and
forskolin further increased by Cact-A1.
Discussion
Screening of ∼110,000 synthetic small molecules and
natural products identified the novel amino-carbonitrilepyrazole CFTR activator Cact-A1, which activated WT- and
ΔF508-CFTR Cl2 currents without cAMP elevation and without the need for a cAMP agonist. Though Cact-A1 produced
linear Cl2 CFTR currents, its activity in the absence of a
cAMP agonist, in contrast to existing activators and potentiators
such as genistein and VX-770, suggests a novel mechanism
of action. Cact-A1 or other CFTR “activators” are potential
alternatives for therapy of CF caused by the ΔF508 mutation
and, potentially, some gating mutations.
Recent analysis of VX-770 activation of CFTR suggests two
distinct mechanisms—an ATP-dependent and an unconventional (ATP-independent) mechanism. VX-770 not only increases the open time of WT-CFTR by stabilizing a posthydrolytic
open state in an ATP-dependent manner, but also increases
the spontaneous ATP-independent opening rate of CFTR
(Eckford et al., 2012; Jih and Hwang, 2013). The binding
site of VX-770 is not known, though there are clues that it
may bind to the transmembrane domains of CFTR (Jih and
Hwang, 2013). The different properties of Cact-A1 versus
VX-770 with regard to CFTR activation in the absence of
a cAMP agonist suggest different mechanisms of action.
cAMP agonists activate CFTR by phosphorylation of the its
regulatory domain; however, Cact-A1 does not elevate intracellular cAMP. As shown in Fig. 3C, Cact-A1 competes with
CFTRinh-172, a CFTR-selective inhibitor, and a high concentration of VX-770 (30 mM) produced little CFTR Cl2 current,
whereas Cact-A1 strongly activated CFTR Cl2 current without
forskolin (Fig. 4D). Further, the additive effects of Cact-A1 and
forskolin for ΔF508-CFTR (Fig. 5A) suggest direct interaction
between CFTR and Cact-A1, as well as an unconventional
mechanism of CFTR activation.
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 6. G551D-CFTR is weakly activated but
strongly potentiated by Cact-A1 in G551D-CFTR
expressing FRT cells. (A) G551D-CFTR was
stimulated by application of 30 mM Cact-A1,
20 mM forskolin, and 10 mM VX-770. G551DCFTR-dependent current was inhibited by 20 mM
CFTRinh-172. (B) Summary of peak currents
(mean 6 S.E., n = 4–7). (C) Whole-cell G551DCFTR currents were recorded at a holding potential at 0 mV, and pulsing to voltages between
680 mV (in steps of 20 mV) in G551D-CFTRexpressing FRT cells. Bottom right: summary of
current density data measured at 180 mV (mean 6
S.E., n = 4–7). *P , 0.05; **P , 0.001.
Novel Small-Molecule CFTR Activator
391
Because Cact-A1 can activate CFTR in the absence of cAMP
agonists, there are potential concerns about in vivo side
effects, such as increased intestinal fluid secretion and diarrhea. However, in CF patients with loss of function CFTR
mutations, inappropriate overactivation of CFTR by Cact-A1
is unlikely.
Current results suggest that drug therapy of CF caused by the
ΔF508 mutation will require a corrector to rescue ΔF508-CFTR
cell surface expression, and a potentiator to increase its Cl2
conductance. Recent clinical data using the corrector VX-809
(3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) in CF patients
with DF508 mutation suggest that it will not be sufficient to
produce a clinical benefit (Van Goor et al., 2011; Clancy et al.,
2012). VX-809 is currently in phase 2 clinical trials using
combination treatment with VX-770. The addition of a potentiator to maximize cell Cl2 conductance is the accepted current
concept in CF therapeutics development. Alternatively, an efficient corrector or combination of correctors that restore proper
ΔF508-CFTR folding and plasma membrane targeting may
obviate the need for a potentiator. The combination of a corrector
and an activator such as Cact-A1 here may be beneficial to
maximize ΔF508-CFTR Cl2 conductance, even in the absence
of cAMP elevation.
In summary, Cact-A1, a novel CFTR activator, activated CFTR
Cl2 current without cAMP elevation and was CFTR-selective,
reversible, and nontoxic. In primary cultures of human airway
epithelial cells, Cact-A1 strongly stimulated WT- and DF508CFTR to the same level as that produced by forskolin, and
showed additive activation of DF508-CFTR with forskolin.
Cact-A1 may be useful for elucidating molecular mechanisms
of CFTR activation and as a potential CF drug development
candidate.
Authorship Contributions
Participated in research design: Namkung, Verkman.
Conducted experiments: Namkung, Park, Seo.
Contributed new reagents or analytic tools: Namkung.
Performed data analysis: Namkung.
Wrote or contributed to the writing of the manuscript: Namkung,
Verkman.
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